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RecA: Week 5, June 13-18
From 2011.igem.org
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==Wednesday== | ==Wednesday== | ||
===RecAI Mutagenesis Test, Take 2 Day 8=== | ===RecAI Mutagenesis Test, Take 2 Day 8=== | ||
| - | The plates featuring cells containing the assembly from 6/14 contained no colonies. The PcI reporter ( | + | The plates featuring cells containing the assembly from 6/14 contained no colonies. The PcI reporter (I763007) and the terminator (B0015) pieces from the registry were transformed into Escherichia coli cells and were left in culture overnight. |
===RecAI Extraction Take 4, Day 2=== | ===RecAI Extraction Take 4, Day 2=== | ||
The only colonies that grew overnight were red, indicating that the RFP gene was not replaced by the RecAI gene. Thus, the restriction digests made on 6/14 were ligated then transformed into Escherichia coli cells and plated onto kanamycin plates. The purified RecAI PCR products were sent to sequencing to ensure that the RecAI extraction worked. | The only colonies that grew overnight were red, indicating that the RFP gene was not replaced by the RecAI gene. Thus, the restriction digests made on 6/14 were ligated then transformed into Escherichia coli cells and plated onto kanamycin plates. The purified RecAI PCR products were sent to sequencing to ensure that the RecAI extraction worked. | ||
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| + | ==Thursday== | ||
| + | ===RecAI Mutagenesis Test, Take 2 Day 9=== | ||
| + | DNA from the cultures created on 6/15 containing the terminator (B0015) and the PcI reporter (I763007) was extracted through the Omega Bio-Tek miniprep protocol. The PcI reporter insert was amplified through PCR, and the product was purified. Since the registry DNA containing the PcI reporter and the terminator parts was running low, freezer stock was made of the two parts for both the -80°C and the -20°C freezers. | ||
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| + | One proposed hypothesis for the continuous failed assemblies was the restriction enzymes have died. For this assembly, XbaI and SpeI enzymes from Dr. Salis' lab were used for restriction digest. SpeI was used instead of PstI because Dr. Salis did not have PstI in his lab. Dr. Salis also suggested doing longer restriction digest (4-5 hours instead of 1-2 hours) in order to obtain more DNA. | ||
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[[Team:Penn_State/Notebook| Back to Notebook]] | [[Team:Penn_State/Notebook| Back to Notebook]] | ||
Revision as of 18:07, 23 June 2011
Contents |
Monday
RecAI Mutagenesis Test, Take 2 Day 6
No colonies grew over the weekend. Thus, the PcI reporter (I763007) was assembled to the terminator (B0015) through amplified insert assembly. The assembly hit a road block at the plating stage because no agar plates existed in the lab. Thus, the RecA group attempted to make plates themselves. The transformed cells were grown in culture, but the recovery media with the cells were kept in order to plate the next day, once more plates were made.
The testing construct will be able to use either part K648001 or K648002 parts, which were assembled and sequenced by the sensor group. These parts feature the constitutive promoter in front of the cI represser, which is necessary for the RecA1 mutagenesis testing construct.
Tuesday
RecAI Mutagenesis Test, Take 2 Day 7
The assembly from 6/13 was plated early in the morning in order for the colonies to grow all day and be ready for colony PCR at night. Unfortunately, the colonies did not grow again.
RecAI Extraction Take 4, Day 1
In order to store the RecAI mutant strain in a plasmid, the RecAI part was inserted into the K3 vector since the C3 vector did not work. Ampified insert assembly was used for this insertion. The RecAI PCR product was purified and digested with XbaI and PstI. The K3 miniprep from 6/8 was digested with XbaI and PstI. The two digests were ligated together, and the ligation was transformed into Esherichia coli cells and plated onto kanamycin resistant plates.
Wednesday
RecAI Mutagenesis Test, Take 2 Day 8
The plates featuring cells containing the assembly from 6/14 contained no colonies. The PcI reporter (I763007) and the terminator (B0015) pieces from the registry were transformed into Escherichia coli cells and were left in culture overnight.
RecAI Extraction Take 4, Day 2
The only colonies that grew overnight were red, indicating that the RFP gene was not replaced by the RecAI gene. Thus, the restriction digests made on 6/14 were ligated then transformed into Escherichia coli cells and plated onto kanamycin plates. The purified RecAI PCR products were sent to sequencing to ensure that the RecAI extraction worked.
Thursday
RecAI Mutagenesis Test, Take 2 Day 9
DNA from the cultures created on 6/15 containing the terminator (B0015) and the PcI reporter (I763007) was extracted through the Omega Bio-Tek miniprep protocol. The PcI reporter insert was amplified through PCR, and the product was purified. Since the registry DNA containing the PcI reporter and the terminator parts was running low, freezer stock was made of the two parts for both the -80°C and the -20°C freezers.
One proposed hypothesis for the continuous failed assemblies was the restriction enzymes have died. For this assembly, XbaI and SpeI enzymes from Dr. Salis' lab were used for restriction digest. SpeI was used instead of PstI because Dr. Salis did not have PstI in his lab. Dr. Salis also suggested doing longer restriction digest (4-5 hours instead of 1-2 hours) in order to obtain more DNA.
